The laboratory technique of Polymerase Chain Reaction (PCR) will by now be well known, at least in principle, to most people from its extensive use in testing for a particular virus. In very simple terms, PCR is a method of amplifying a DNA target sequence through repeated heating and cooling cycles that amplify the target sequence until it reaches a level high enough to be detected. PCR can therefore be used to detect extremely small amounts of a particular sequence, provided enough cycles are used. As the inventor Kary Mullis put it, "you can find almost anything in anybody" (see here). How many cycles are used is therefore critical to how much reliance you put on the results, particularly if you are using it to diagnose the presence of a virus (the RNA sequences being converted to DNA for RT-PCR testing).
To have confidence in the results, a PCR machine will need calibrating and validating to ensure that the thermal cycling is accurately and repeatedly carried out and that the amount detected after each cycle is accurate. If this is done properly, the technique can be used to establish quantitatively as well as qualitatively what was present in the original sample, i.e. not just whether something was there but also how much.
Fluorescent dyes can be used that allow measurements to be made that correlate the amount of the detected substance with a fluorescent response, which allows an instrument to be calibrated and validated so that it can be known how a measured response corresponds to an amount of material present. Such dyes might, for example, be used in the form of a standard solution put in a PCR machine to establish a calibrated level.
GB patent application 1421819.2 disclosed the use instead of a fluorophore suspended in a thermoplastic polymer matrix, for use in PCR validation. A PCR tube made of the polymer (shown here on the right, the sole drawing in the application) with a fluorophore suspended in the polymer, allowed a spectrometer to detect a known level of fluorescence so that the PCR machine could be validated. Claim 1 of the application as filed read:
The examiner objected that the claimed invention lacked an inventive step, given that suspended fluorophores were known and the choice of polymer was obvious. The applicant was unable to get over this and the issue came before a hearing officer at the UK IPO. By this stage, claim 1 had been amended to add a few more features:
1. Use of a fluorophore suspended in a thermoplastic polymer matrix, which fluorophore suspended in a thermoplastic polymer matrix has fluorescence characteristics within a predetermined uncertainty budget, wherein the polymer matrix is selected from the group consisting of: poly methyl methacrylate (PMMA), polycarbonate (PC), polyoxymethylene (POM), chlorinated polyvinyl chloride (CPVC) or PVC/Acrylic copolymer to validate a thermal cycler.
1. A method of validating a thermal cycler, characterised by the steps of:
suspending a fluorophore in a thermoplastic polymer matrix, wherein the fluorophore has fluorescence characteristics within a predetermined uncertainty budget, and wherein the polymer matrix is selected from the group consisting of: methyl methacrylate (PMMA), polycarbonate (PC), polyoxymethylene (POM), chlorinated polyvinyl Chloride (CPVC) Or PVC/Acrylic copolymer, and the thermoplastic matrix is cut to the shape of a PCR tube; and
validating the thermal cycler using measurements obtained in the thermal cycler and the predetermined uncertainty of the fluorophore:wherein validating the thermal cycler includes confirming that the measurements obtained the thermal cycler match the fluorescence characteristics of the fluorophore within the predetermined uncertainty budget.